The Alphaviruses are enveloped, plus strand RNA viruses within the Togaviridae family, and are important human and animal pathogens. These viruses invade the host cells through the receptor-mediated endocytosis pathway. The acidic environment in the endosome induces fusion of the viral and endosomal membranes, allowing delivery of the viral genome into the cytoplasm of the infected cell. The energy cost for merging the hydrated membranes is overcome by the conformational changes and oligomeric rearrangements of the viral E1 glycoproteins, a class-II fusion protein. Although the crystal structures of the E1 ecto-domain in pre- and post-fusion conformations were determined, the structural details of the intermediate organizations of the fusion protein during the course of membrane fusion are poorly understood. Major obstacles that have impeded vigorous structural studies are the aggregation and heterogeneity of virus particles at low-pH, and additional heterogeneity introduced by nonuniform target membranes. The proposed research employs several novel procedures to overcome such difficulties and produce eligible samples of Alphavirus in complex with lipid membranes at acidic pH. Cryo-electron microscopy and three-dimensional image reconstruction techniques will be used to determine the structure of the virus-membrane complex at nanometer resolution. Fitting the crystal structures of the E1 protein into the reconstruction map will lead to discovery of the conformation, oligomerization and organization of E1 when it inserts into a target membrane upon acidification. The long-term objective of the proposed research is to outline the detailed steps of viral protein conformational changes correlating with membrane remodeling and fusion. The experience obtained from this study can be generalized and applied to similar structural investigations that involve other small-enveloped viruses, such as Flaviviruses. Knowledge gained in this study will also be beneficial for the development of antiviral strategies. PUBLIC HEALTH RELEVANCE: A key step in the infection of enveloped viruses, such as influenza virus, West Nile virus and Sindbis virus, into the host cell, is fusion of the virus envelope with the host cell membrane. This research aims to illustrate the structural details of this process driven by the Sindbis viral fusion proteins, and provides a general approach for structural investigation of membrane fusion carried out by the enveloped viruses. Knowledge gained from such studies provides potential targets for anti-viral reagents.